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Research Article
Is the mazEF toxin-antitoxin system responsible for vancomycin resistance in clinical isolates of Enterococcus faecalis? Ist das mazEF Toxin-Antitoxin-System verantwortlich für die Vancomycinresistenz klinischer Isolate von Enterococcus faecalis? Abstract The current study was conducted to investigate the relationship between vancomycin-resistant Enterococcus faecalis (VRE) and the presence of mazEF toxin-antitoxin (TA) system, which may be useful as target for novel antimicrobial therapy concepts. The susceptibility of E. faecalis was determined by MIC, and the presence of the mazEF TA system was evaluated by PCR. Among 200 E. faecalis isolates 39.5% showed resistance to vancomycin (VRE), while 60.5% were susceptible strains (VSE). The mazEF TA system was positive in all VRE isolates (100%), but less prevalent (38/121, 31.4%) among the 121 VSE strains. In conclusion, our study demonstrated a positive relationship between the presence of vancomycin resistance and mazEF TA system. This observation may introduce therapeutic options against a novel antimicrobial target in enterococci.
Nourkhoda Sadeghifard1 Sara Soheili1 Zamberi Sekawi1 Sobhan Ghafourian1 1 Department of Medical Microbiology, University Putra Malaysia, Malaysia
Keywords: Enterococcus, mazEF TA system, vancomycin, antimicrobial target
Zusammenfassung In der vorliegenden Studie sollte der Zusammenhang zwischen der Vancomycinresistenz bei Enterococcus faecalis (VRE) und dem Vorkommen des mazEF Toxin-Antitoxin(TA)-Systems untersucht werden, um Hinweise für eine neue antimikrobielle Targettherapie zu erhalten. Die Empfindlichkeit der Enterkokokken wurde mittels MIC, das Vorkommen des mazEF TA-Systems mittels PCR bestimmt. Unter 200 Enterokokkenisolaten waren 39,5% resistent gegen Vancomycin (VRE), während 60,5% sensibel waren. Das mazEF TA-System war bei allen VRE-Isolaten positiv, aber weniger prävalent bei den VSEIsolaten (38/121, 31,4%). Die Analyse ergab einen Zusammenhang zwischen dem Vorkommen des VRE-Resistenzgens und dem mazEF TA-System, was für weitere Untersuchungen in Hinblick auf ein neues antimikrobielles Ziel gegenüber Enterokokken hilfreich ist. Schlüsselwörter: Enterococcus, mazEF AT-System, Vancomycin, antimikrobielles Target
Introduction Enterococcus is one of the important organisms which is responsible for clinical infections such as urinary tract infections, bacteremia, bacterial endocarditis, diverticulitis, and meningitis [1]. Enterococcus antibiotic sensitive strains can be treated with β-lactam antibiotics such as
ampicillin and glycopeptides such as vancomycin [2]. Regretfully, resistance against antibiotics is increasing globally [3]. In the last two decades, vancomycin resistance of enterococci has emerged increasingly in nosocomial infections of hospitalized patients [1]. Mobile genetic elements are responsible for the vancomycin resistance in enterococci [4]. Despite the frequency
GMS Hygiene and Infection Control 2014, Vol. 9(1), ISSN 2196-5226
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Sadeghifard et al.: Is the mazEF toxin-antitoxin system responsible for ...
of plasmid resistance among vancomycin-resistant enterococci (VRE) isolates is not obvious, there are multiple reports of plasmids with the various vancomycin resistant genes clusters. Some plasmids harbor loci encoded postsegregation killing systems [5], which encode a toxin and its corresponding antitoxin. The analysis showed that this system could be vertically transferred. The antitoxin is unstable; however, the toxin is stable, which adds to postsegregation killing systems [6]. Interestingly, the stable toxin may trigger bacteriostasis among VRE strains. The current study was conducted to explain the relationship between vancomycin resistance of Enterococcus faecalis and the presence of mazEF TA system, which may be useful as target for novel antimicrobial therapy concepts.
step in 72°C for 10 minutes. Then, the PCR products were analyzed by 1% agarose gel electrophoresis.
Results Prevalence of VRE Among the 200 E. faecalis isolates (Table 1), 39.5% of the isolates showed resistance to vancomycin (VRE), while 60.5% were susceptible to vancomycin (VSE) (Figure 1). Table 1: Prevalence of VRE in Milad and Ilam Hospitals
Methods Bacterial isolates and identification Two hundred isolates of Enterococcus faecalis were collected during September 2011 and April 2012 in Ilam Hospital in the West of Iran and Milad Hospital in Tehran, the capital of Iran. The isolates were obtained from patients with urinary tract infection. The Enterococcus faecalis identification was performed by Gram staining, motility assessment, catalase production, growth in 6.5% NaCl, xylose, mannitol, arabinose, sorbitol use, bile, and esculin growth. Strains were additionally tested for hydrolysis, pigment production, leucine aminopeptidase activity, and acidification of methyl-a-D-glucopyranoside [7].
Determination of vancomycin-resistant Enterococcus (VRE) Minimum inhibitory concentrations (MICs) were assessed by microdilution in Mueller-Hinton broth. MICs of vancomycin were defined as being resistant to vancomycin with an MIC ≥8 µg/mL [8]. E. faecalis ATCC 51299 was used as positive control [8].
Determination of the MazEF TA system The specific primers were designed for mazEF TA loci to amplify 505 bp oligonucleotide. The primer sequences of PCR primers were as belows: Forward: 5-ATGATCCACAGTAGCGTAAAGCGT-3; Reverse: 5-TTACCAGACTTCCTTATCTTTCGG-3. The PCR amplification was carried out in a final volume of 25 µl with 3 µl of DNA as a template, 2.5 µl PCR buffer (20 mM Tris-HCl/50 mM KCl, pH 8.4), 1.5 mM MgCl2, 1 mM each deoxynucleoside triphosphate, 1 µM each primer, and 2 units of Taq polymerase. The PCR was performed with an initial denaturation in 95°C for 2 minutes and 35 cycles of denaturation in 94°C for 1 minute, annealing in 58°C for 45 seconds, and extension in 72°C for 30 seconds, following in a final extension
Figure 1: PCR analysis of VRE clinical isolates; M (Marker 1 kb plus, Thermo Science), 1 = mazEF, 505 bp)
Presence of the MazEF TA system All 200 E. faecalis strains were analyzed by PCR, and findings were designated as positive if a distinct band was found at the expected size on an agarose gel. The PCR results showed that the mazEF TA system was found in all VRE isolates (79/79, 100%), however, only in 31.4% (n=38) of the 121 VSE isolates. This difference was statistically significant (p
Research Article
Is the mazEF toxin-antitoxin system responsible for vancomycin resistance in clinical isolates of Enterococcus faecalis? Ist das mazEF Toxin-Antitoxin-System verantwortlich für die Vancomycinresistenz klinischer Isolate von Enterococcus faecalis? Abstract The current study was conducted to investigate the relationship between vancomycin-resistant Enterococcus faecalis (VRE) and the presence of mazEF toxin-antitoxin (TA) system, which may be useful as target for novel antimicrobial therapy concepts. The susceptibility of E. faecalis was determined by MIC, and the presence of the mazEF TA system was evaluated by PCR. Among 200 E. faecalis isolates 39.5% showed resistance to vancomycin (VRE), while 60.5% were susceptible strains (VSE). The mazEF TA system was positive in all VRE isolates (100%), but less prevalent (38/121, 31.4%) among the 121 VSE strains. In conclusion, our study demonstrated a positive relationship between the presence of vancomycin resistance and mazEF TA system. This observation may introduce therapeutic options against a novel antimicrobial target in enterococci.
Nourkhoda Sadeghifard1 Sara Soheili1 Zamberi Sekawi1 Sobhan Ghafourian1 1 Department of Medical Microbiology, University Putra Malaysia, Malaysia
Keywords: Enterococcus, mazEF TA system, vancomycin, antimicrobial target
Zusammenfassung In der vorliegenden Studie sollte der Zusammenhang zwischen der Vancomycinresistenz bei Enterococcus faecalis (VRE) und dem Vorkommen des mazEF Toxin-Antitoxin(TA)-Systems untersucht werden, um Hinweise für eine neue antimikrobielle Targettherapie zu erhalten. Die Empfindlichkeit der Enterkokokken wurde mittels MIC, das Vorkommen des mazEF TA-Systems mittels PCR bestimmt. Unter 200 Enterokokkenisolaten waren 39,5% resistent gegen Vancomycin (VRE), während 60,5% sensibel waren. Das mazEF TA-System war bei allen VRE-Isolaten positiv, aber weniger prävalent bei den VSEIsolaten (38/121, 31,4%). Die Analyse ergab einen Zusammenhang zwischen dem Vorkommen des VRE-Resistenzgens und dem mazEF TA-System, was für weitere Untersuchungen in Hinblick auf ein neues antimikrobielles Ziel gegenüber Enterokokken hilfreich ist. Schlüsselwörter: Enterococcus, mazEF AT-System, Vancomycin, antimikrobielles Target
Introduction Enterococcus is one of the important organisms which is responsible for clinical infections such as urinary tract infections, bacteremia, bacterial endocarditis, diverticulitis, and meningitis [1]. Enterococcus antibiotic sensitive strains can be treated with β-lactam antibiotics such as
ampicillin and glycopeptides such as vancomycin [2]. Regretfully, resistance against antibiotics is increasing globally [3]. In the last two decades, vancomycin resistance of enterococci has emerged increasingly in nosocomial infections of hospitalized patients [1]. Mobile genetic elements are responsible for the vancomycin resistance in enterococci [4]. Despite the frequency
GMS Hygiene and Infection Control 2014, Vol. 9(1), ISSN 2196-5226
1/4
Sadeghifard et al.: Is the mazEF toxin-antitoxin system responsible for ...
of plasmid resistance among vancomycin-resistant enterococci (VRE) isolates is not obvious, there are multiple reports of plasmids with the various vancomycin resistant genes clusters. Some plasmids harbor loci encoded postsegregation killing systems [5], which encode a toxin and its corresponding antitoxin. The analysis showed that this system could be vertically transferred. The antitoxin is unstable; however, the toxin is stable, which adds to postsegregation killing systems [6]. Interestingly, the stable toxin may trigger bacteriostasis among VRE strains. The current study was conducted to explain the relationship between vancomycin resistance of Enterococcus faecalis and the presence of mazEF TA system, which may be useful as target for novel antimicrobial therapy concepts.
step in 72°C for 10 minutes. Then, the PCR products were analyzed by 1% agarose gel electrophoresis.
Results Prevalence of VRE Among the 200 E. faecalis isolates (Table 1), 39.5% of the isolates showed resistance to vancomycin (VRE), while 60.5% were susceptible to vancomycin (VSE) (Figure 1). Table 1: Prevalence of VRE in Milad and Ilam Hospitals
Methods Bacterial isolates and identification Two hundred isolates of Enterococcus faecalis were collected during September 2011 and April 2012 in Ilam Hospital in the West of Iran and Milad Hospital in Tehran, the capital of Iran. The isolates were obtained from patients with urinary tract infection. The Enterococcus faecalis identification was performed by Gram staining, motility assessment, catalase production, growth in 6.5% NaCl, xylose, mannitol, arabinose, sorbitol use, bile, and esculin growth. Strains were additionally tested for hydrolysis, pigment production, leucine aminopeptidase activity, and acidification of methyl-a-D-glucopyranoside [7].
Determination of vancomycin-resistant Enterococcus (VRE) Minimum inhibitory concentrations (MICs) were assessed by microdilution in Mueller-Hinton broth. MICs of vancomycin were defined as being resistant to vancomycin with an MIC ≥8 µg/mL [8]. E. faecalis ATCC 51299 was used as positive control [8].
Determination of the MazEF TA system The specific primers were designed for mazEF TA loci to amplify 505 bp oligonucleotide. The primer sequences of PCR primers were as belows: Forward: 5-ATGATCCACAGTAGCGTAAAGCGT-3; Reverse: 5-TTACCAGACTTCCTTATCTTTCGG-3. The PCR amplification was carried out in a final volume of 25 µl with 3 µl of DNA as a template, 2.5 µl PCR buffer (20 mM Tris-HCl/50 mM KCl, pH 8.4), 1.5 mM MgCl2, 1 mM each deoxynucleoside triphosphate, 1 µM each primer, and 2 units of Taq polymerase. The PCR was performed with an initial denaturation in 95°C for 2 minutes and 35 cycles of denaturation in 94°C for 1 minute, annealing in 58°C for 45 seconds, and extension in 72°C for 30 seconds, following in a final extension
Figure 1: PCR analysis of VRE clinical isolates; M (Marker 1 kb plus, Thermo Science), 1 = mazEF, 505 bp)
Presence of the MazEF TA system All 200 E. faecalis strains were analyzed by PCR, and findings were designated as positive if a distinct band was found at the expected size on an agarose gel. The PCR results showed that the mazEF TA system was found in all VRE isolates (79/79, 100%), however, only in 31.4% (n=38) of the 121 VSE isolates. This difference was statistically significant (p
